Safe and effective approaches for the treatment of obesity are needed to address the tremendous burdens of this disease such as diabetes, cardiovascular disease, and even some cancers. Although studied as distinct pathways for decades, processes regulating eating and motivation are now thought to be driven by overlapping neural circuits. Studying this overlap may provide important insights into the basis of excess caloric intake and lead to the identification of novel pharmacological targets for treating metabolic diseases, the long-term goal of our research. A potential novel and innovative target in this regard is neuromedin U (NMU), a peptide shown to suppress food intake and cause weight loss. These effects of NMU are related to its actions at the NMU receptor 2 (NMUR2) in the hypothalamus, particularly in the paraventricular nucleus (PVN), which is enriched for NMUR2. We have previously found that selective depletion of NMUR2 in the PVN of rats potentiates the intake of a high-fat diet, increases weight gain, and enhances a dietary preference specifically for fat but not sucrose. The goals of the present project are to explore NMUR2 signaling as a key interface between the hypothalamic and mesolimbic systems, and to further evaluate the action of NMUR2 agonists on feeding behavior. Our preliminary data indicate that NMUR2 neurons in the hypothalamus are directly connected to the nucleus accumbens, a key region of the mesolimbic system that regulates reward and motivated behavior. We have, for the first time, evaluated small-molecule NMUR2 agonists in vivo. Our preliminary data indicate that these compounds suppress feeding on a high-fat diet. To accomplish our goals, our Specific Aims are 1) To evaluate the structural link between the hypothalamic and mesolimbic circuits, 2) To establish a functional link between these areas involving enkephalins and opioid signaling, and 3) To determine if NMUR2 is a target to inhibit motivation for high-fat food and regulation of metabolic physiology. These aims will be achieved using such innovative technologies as viral-vector tracers to map neural pathways in the brain, optical clearing with CLARITY visualize these pathways in clarified, intact brains, and virus-mediated RNA interference study the effects of the NMUR2 gene on food intake, body weight, and motivation for food. Overall, these studies will lay the foundation for understanding neural pathways that regulate motivation for food, and for future work developing NMUR2-based therapeutics for the treatment of obesity.